U.S. patent application number 14/551050 was filed with the patent office on 2015-05-28 for high performance liquid chromatography with uv-visible detection.
The applicant listed for this patent is Academia Sinica. Invention is credited to Chung-Hsuan Chen, Chen-Yu Hsieh, Jung-Lee Lin.
Application Number | 20150143880 14/551050 |
Document ID | / |
Family ID | 53181507 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150143880 |
Kind Code |
A1 |
Chen; Chung-Hsuan ; et
al. |
May 28, 2015 |
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH UV-VISIBLE
DETECTION
Abstract
Apparatus and methods for high performance liquid
chromatography. The apparatus includes a preparation loop
comprising two linear stepping pumps, a sample loop comprising a
sample injector, a chromatography column, and a detector device.
The detector device can include a flash lamp, a flow cell, and a
light sensor comprising an entrance slit, a grating; and a
charge-coupled device array.
Inventors: |
Chen; Chung-Hsuan; (Taipei,
TW) ; Hsieh; Chen-Yu; (Taipei, TW) ; Lin;
Jung-Lee; (Taipei, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
|
TW |
|
|
Family ID: |
53181507 |
Appl. No.: |
14/551050 |
Filed: |
November 23, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61908159 |
Nov 24, 2013 |
|
|
|
Current U.S.
Class: |
73/61.58 |
Current CPC
Class: |
G01N 2030/027 20130101;
G01N 21/33 20130101; G01N 21/33 20130101; G01N 30/74 20130101; G01N
30/74 20130101 |
Class at
Publication: |
73/61.58 |
International
Class: |
G01N 30/74 20060101
G01N030/74 |
Claims
1. A liquid chromatography apparatus comprising: a preparation loop
comprising two linear stepping pumps; a sample loop comprising a
sample injector; a chromatography column; and a detector device
comprising: a flash lamp: a flow cell; and a light sensor
comprising: an entrance slit; a grating; and a charge-coupled
device array.
2. The apparatus of claim 1, wherein the grating is an etched
silicon light diffraction grating having convex shape for focusing
diffracted light.
3. The apparatus of claim 1, wherein the chromatography column is
selected from an adsorption, a partition, an ion exchange, an
affinity, and a size exclusion retention column.
4. The apparatus of claim 1, wherein the preparation loop can be
operated in either isocratic or differential pumping modes.
5. The apparatus of claim 1, wherein the detector device has a scan
range of from 230 nm to 800 nm.
6. The apparatus of claim 1, wherein the light sensor is a
UV-Visible light sensor.
7. The apparatus of claim 1, wherein the stepping pumps have a
minimum step of 0.8 nanoliter.
8. The apparatus of claim 1, wherein the flash lamp is a
xenon-based flash lamp.
9. The apparatus of claim 1, wherein the flash lamp is operated
with a duty cycle of 50% or less at a power of 2 Watt.
10. The apparatus of claim 1, further comprising a microcomputer
controller configured to synchronize the operation of the flash
lamp to the operation of the charge-coupled device array.
11. The apparatus of claim 1, wherein the flow cell is an HPLC flow
cell.
12. The apparatus of claim 1, wherein the liquid chromatography
apparatus has a total volume of less than 10 L.
13. A method for performing liquid chromatography, the method
comprising: providing analytes in a carrier liquid; step-pumping
the carrier liquid through a chromatography column to separate the
analytes; detecting the separated analytes with a detector device
comprising a flash lamp and a UV-Visible light sensor comprising a
grating and a charge-coupled device array.
14. The method of claim 13, wherein the grating is an etched
silicon light diffraction grating having convex shape for focusing
diffracted light.
15. The method of claim 13, wherein the chromatography column is
selected from an adsorption, a partition, an ion exchange, an
affinity, and a size exclusion retention column.
16. The method of claim 13, wherein the detector device has a scan
range of from 230 nm to 800 nm.
17. The method of claim 13, wherein the step-pumping has a minimum
step of 0.8 nanoliter.
18. The method of claim 13, wherein the flash lamp is operated at a
power of 2 Watt.
19. The method of claim 13, further comprising operating a
microcomputer controller to synchronize the operation of the flash
lamp to the operation of the charge-coupled device array.
20. The method of claim 13, further comprising focusing diffracted
light from the grating onto the charge-coupled device array.
Description
BACKGROUND OF THE INVENTION
[0001] High performance liquid chromatography (HPLC) is one of the
most important analytical techniques. HPLC techniques are widely
applied in chemistry, medical and pharmaceutical uses,
environmental and food sciences, and many other fields.
[0002] Liquid chromatography employs a liquid as the carrier for
analyte samples. The carrier or mobile phase passes through a
stationary phase composed of particles packed in a tube or column.
In a conventional HPLC, the particles can be about 7 um in
size.
[0003] HPLC can be applied to analyte mixtures or species which can
be dissolved in water, as well as polymers dissolved in organic
solvents.
[0004] Recently, new HPLC-related techniques have been developed
with improved means of detection. For example, Ultrahigh Pressure
Liquid Chromatography (UPLC), Rapid Resolution Liquid
Chromatography (RRLC), Ultrafast Liquid Chromatography (UFLC),
Rapid Separation Liquid Chromatography (RSLC), and Fast Protein
Liquid Chromatography (FPLC).
[0005] A drawback of conventional liquid chromatography methods and
systems is the difficulty in combining the liquid chromatography
apparatus with a mass spectrometer apparatus or other large,
conventional analyte detection systems. A liquid chromatography
apparatus having a large mass and size is difficult to use in
combination with a mass spectrometer that is also typically
large.
[0006] Further, in medical and health care settings, it would be
desirable to have a small and efficient liquid chromatography
apparatus so that certain measurements can be done on-site in a
patient clinic, rather than being carried out in a larger facility
such as a medical center or hospital.
[0007] Moreover, for environmental measurements that should be
performed in the field or at remote locations such as a rainforest,
a geological location such as a volcano, or in a spacecraft or
extraterrestrial location, it can be required to have a small and
efficient liquid chromatography apparatus. Such a device would
allow detection of analytes, chemicals, contaminants or other
species at their source.
[0008] Another drawback of conventional liquid chromatography
methods and systems is the use of a photomultiplier as a light
sensor. When a photomultiplier is used as a light sensor, a
monochromator and mirrors are generally required to isolate
wavelengths of light from the sample.
[0009] On the other hand, it is desirable to have a liquid
chromatography apparatus that can simultaneously and efficiently
detect a range of wavelengths of light.
[0010] A further drawback of conventional liquid chromatography
methods and systems is in using screw-operated pump heads for
liquid transport, which have large mass.
[0011] There is a continuing need for a liquid chromatography
apparatus having a detector device that can measure a range of
wavelengths of light simultaneously. There is also a need for a
liquid chromatography apparatus that can provide efficient pumping
with less mass. And there is a long standing need for efficient
liquid chromatography methods and systems for use with other
detection systems, as well as for making measurements in the
field.
BRIEF SUMMARY
[0012] This invention relates to the fields of liquid
chromatography and analyte detection. More particularly, this
invention relates to methods and devices for high performance
liquid chromatography for efficiently generating chromatograms of
analytes with UV-visible and other methods of detection, as well as
for reducing the mass requirements of a liquid chromatography
apparatus to enhance utilization in several fields.
[0013] This invention provides methods and devices for high
performance liquid chromatography for efficiently generating
chromatograms of analytes with UV-visible and other methods of
detection, as well as for reducing the mass requirements of a
liquid chromatography apparatus to enhance utilization in several
fields.
[0014] Embodiments of this invention include a liquid
chromatography apparatus having a detector device that can measure
a range of wavelengths of light simultaneously. A liquid
chromatography apparatus of this invention can provide efficient
pumping with less mass.
[0015] Embodiments of this invention include:
[0016] A liquid chromatography apparatus comprising: [0017] a
preparation loop comprising two linear stepping pumps; [0018] a
sample loop comprising a sample injector; [0019] a chromatography
column; and [0020] a detector device comprising: [0021] a flash
lamp: [0022] a flow cell; and [0023] a light sensor comprising:
[0024] an entrance slit; [0025] a grating; and [0026] a
charge-coupled device array.
[0027] The apparatus above, wherein the grating is an etched
silicon light diffraction grating having convex shape for focusing
diffracted light.
[0028] The apparatus above, wherein the chromatography column is
selected from an adsorption, a partition, an ion exchange, an
affinity, and a size exclusion retention column.
[0029] The apparatus above, wherein the preparation loop can be
operated in either isocratic or differential pumping modes.
[0030] The apparatus above, wherein the detector device has a scan
range of from 230 nm to 800 nm.
[0031] The apparatus above, wherein the light sensor is a
UV-Visible light sensor.
[0032] The apparatus above, wherein the stepping pumps have a
minimum step of 0.8 nanoliter.
[0033] The apparatus above, wherein the flash lamp is a xenon-based
flash lamp.
[0034] The apparatus above, wherein the flash lamp is operated with
a duty cycle of 50% or less at a power of 2 Watt.
[0035] The apparatus above, further comprising a microcomputer
controller configured to synchronize the operation of the flash
lamp to the operation of the charge-coupled device array.
[0036] The apparatus above, wherein the flow cell is an HPLC flow
cell.
[0037] The apparatus above, wherein the liquid chromatography
apparatus has a total volume of less than 10 L.
[0038] A method for performing liquid chromatography, the method
comprising: [0039] providing analytes in a carrier liquid; [0040]
step-pumping the carrier liquid through a chromatography column to
separate the analytes;
[0041] detecting the separated analytes with a detector device
comprising a flash lamp and a UV-Visible light sensor comprising a
grating and a charge-coupled device array.
[0042] The method above, wherein the grating is an etched silicon
light diffraction grating having convex shape for focusing
diffracted light.
[0043] The method above, wherein the chromatography column is
selected from an adsorption, a partition, an ion exchange, an
affinity, and a size exclusion retention column.
[0044] The method above, wherein the detector device has a scan
range of from 230 nm to 800 nm.
[0045] The method above, wherein the step-pumping has a minimum
step of 0.8 nanoliter.
[0046] The method above, wherein the flash lamp is operated at a
power of 2 Watt.
[0047] The method above, further comprising operating a
microcomputer controller to synchronize the operation of the flash
lamp to the operation of the charge-coupled device array.
[0048] The method above, further comprising focusing diffracted
light from the grating onto the charge-coupled device array.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1: FIG. 1 shows a schematic of an embodiment of a
liquid chromatography apparatus of this invention. The liquid
chromatography apparatus can include two or more linear stepping
pumps 10, pressure transducers 20, a flow meter 30, a solvent mixer
35, an actuated carrier valve 40, an actuated sample valve 50, a
sample injector 60, carrier reservoirs 70, a detector device 80, a
column 90, and a microcomputer controller.
[0050] FIG. 2: FIG. 2 shows a schematic of an embodiment of a
detector device 80 of this invention. The detector device can
include a flash lamp 110, light fibers 120, a flow cell 130, and a
UV-Vis light sensor 140. The UV-Vis light sensor 140 can include an
entrance slit 150, a grating 160, and a CCD array 170.
[0051] FIG. 3: FIG. 3 shows a chromatogram of a sample of MyoGlobin
obtained with an HPLC apparatus of this invention. The chromatogram
shows retention time versus absorbance and was obtained with UV-Vis
absorbance at 400 nm wavelength. The HPLC apparatus of this
invention had a total volume less than 10 L and was operated with
less than 150 Watt overall power consumption.
DETAILED DESCRIPTION OF THE INVENTION
[0052] Embodiments of this invention provide apparatuses and
methods for high performance liquid chromatography. The high
performance liquid chromatography apparatus of this invention can
provide rapid detection and separation of a complex mixture of
analytes.
[0053] An HPLC apparatus of this disclosure can have one or more
pumps, pressure and flow sensors, switch valves, a column used to
separate an analyte mixture, and a detector to analyze samples of
analytes.
[0054] In some embodiments, a liquid chromatography apparatus of
this invention can have one or more pumps, a flow control feedback
system, an analyte column, and a detector device.
[0055] In some embodiments, an analyte column can have many small
particles packed inside forming a stationary phase. Mixtures of
analytes can be separated by passing the mixture in a mobile phase
through the stationary phase of the column. Different analytes will
require different retention times to pass through the column. A
separated or purified sample can be generated at the end of
column.
[0056] Embodiments of a liquid chromatography apparatus of this
invention can employ columns for adsorption, partition, ion
exchange, affinity, and size exclusion retention.
[0057] In some embodiments, a liquid chromatography apparatus of
this invention can have a reservoir for a carrier liquid, and a
chromatography column in fluid communication with the
reservoir.
[0058] Embodiments of this invention include a liquid
chromatography apparatus that can provide a continuous and stable
flow of carrier solvent through the column. The continuous and
stable flow of carrier can provide the ability to separate and
detect individual analytes.
[0059] In certain embodiments, this invention includes a liquid
chromatography apparatus that can provide a continuous and stable
flow of analytes and carrier so that quantitative measurements of
analytes can be performed.
[0060] In some aspects, an HPLC apparatus of this invention can
have a flow control system. A flow control system can have one or
more pressure transducers that feed back to the one or more pumps
to maintain a constant pressure in the column.
[0061] In certain embodiments, a flow control system can have a
flow meter that feeds back to a pump to maintain a constant flow
speed in the column.
[0062] In certain embodiments, an HPLC apparatus of this invention
can operate in either isocratic or differential pumping modes.
[0063] In some embodiments, the carrier solvent can be driven by a
single mechanical pump in isocratic pumping mode. In isocratic
pumping, the composition of the mobile phase may remain
constant.
[0064] In further embodiments, the carrier solvent can be driven by
two mechanical pumps in differential pumping mode. In differential
pumping, the composition of the mobile phase varies during
acquisition of an HPLC chromatogram to improve resolution and
separation. In differential pumping, different solvents can be
pumped and mixed at different speeds.
[0065] In some aspects, the apparatus of this disclosure maintains
a constant pumping speed. A pressure transducer and a flow sensor
can be used to provide mechanical pump feedback to maintain a
constant pumping speed using a microcomputer.
[0066] In certain embodiments, an HPLC apparatus of this invention
can have one or two pump heads each having a single piston. A pump
head can have a sapphire piston and a stainless steel pump
head.
[0067] An example of a single piston pump is an DRL42PB2-04
(Oriental Motors).
[0068] In another aspect, a liquid chromatography apparatus of this
invention can have one or two linear stepping motors to operate the
pump heads. A linear stepping motor advantageously provides more
efficient pumping as compared to a screw-driven stepping motor. The
use of linear stepping motors can reduce power consumption, as well
as reduce the required size of the corresponding pump head and
maintain constant pumping speeds.
[0069] In some embodiments, the pumping step or resolution can be
0.8 nanoliter (nl).
[0070] The mass flow rate of an HPLC of this disclosure can be from
nanoliters/min to microliters/min. In some embodiments, the mass
flow rate of an HPLC of this disclosure can be from 1 nL/min to 20
uL/min.
[0071] The pressure in an HPLC of this disclosure can be from 200
to 500 Bar.
[0072] An HPLC of this disclosure can include an injector which
introduces the sample into the flowing carrier. The volume of a
sample for an HPLC of this disclosure can be from 1 to 20
microliters.
[0073] In some embodiments, this invention provides a
multi-wavelength UV-visible detector device for high performance
liquid chromatography.
[0074] A detector device of this invention can include a flash
lamp, a flash lamp controller, a flow cell, and a UV-Visible light
sensor.
[0075] An example of a flow cell is an HPLC flow cell.
[0076] In some aspects, the flash lamp can be a miniature xenon
based flash lamp (e.g., Excelitas).
[0077] In certain aspects, an apparatus of this invention includes
a lamp controller that synchronizes the illumination of the lamp to
the operation of the light sensor. By synchronizing the
illumination of the lamp to the operation of the light sensor, an
apparatus of this disclosure can provide high light intensity and
reduce the electrical power required to operate the apparatus for
the detection of analytes.
[0078] For example, in a detector device of this disclosure, a
flash lamp can be operated with a pulsed duty cycle of 50% or less.
A lamp controller can be used to ensure that the light sensor
operates only when the lamp is on. This operation can reduce the
lamp power required for obtaining the UV-Vis spectrum of analytes
from about 50 Watt to as low as 2 Watt.
[0079] In some embodiments, the light sensor has an entrance slit,
a CCD array, and a silicon grating.
[0080] In some aspects, the silicon grating can be an etched
silicon surface. The silicon grating can have a convex shape for
providing a flat focal plane to match the plane of the CCD
array.
[0081] In general, a CCD (Charge-coupled device) array can detect
many wavelengths of light at the same time. Among other things, the
small physical size of the light sensor of an apparatus of this
invention allows the physical size of the apparatus to be
reduced.
[0082] Using a CCD detector advantageously allows acquisition of a
wide range of wavelengths at the same time without changing the
angle of the grating.
[0083] In some embodiments, detector overall volume and detector
overall power consumption can each be reduced by at least a factor
of ten-fold.
[0084] In further aspects, an HPLC apparatus of this invention can
include a lamp controller microcomputer. The lamp source will in
general operate in pulsed mode. Using the lamp controller, the
excitation light from the lamp can be synchronized to the data
acquisition of the light sensor. By synchronizing the data
acquisition, data is acquired only when the lamp is on during a
flash process.
[0085] By synchronizing the emission of the flash lamp to the light
sensor detecting period, an apparatus of this invention
advantageously provides the high peak power emission of the flash
lamp for detecting analytes.
[0086] Referring to FIG. 1, a liquid chromatography apparatus of
this invention can include two or more pumps 10, pressure
transducers 20, a flow meter 30, a solvent mixer 35, actuated
carrier valve 40, actuated sample valve 50, a sample injector 60,
carrier reservoirs 70, a detector device 80, a column 90, and a
microcomputer controller.
[0087] An embodiment of a liquid chromatography apparatus of this
invention can include two linear stepping pumps 10 to create pump
head pressure on the carrier solvent. By using two stepping pumps,
the pump system can be operated in isocratic or differential
pumping modes.
[0088] The flow system of carrier tubes is used to transfer, mix,
and purge carrier liquids, as well as to provide feedback to the
pump system. The feedback to the pumps can be provided by pressure
transducers 20 and a flow meter 30. The feedback can be used to
control the carrier flow speed and pressure. The feedback control
can maintain a steady, continuous carrier flow speed and
pressure.
[0089] The flow system of carrier tubes can be placed in fluid
communication using multi-direction actuated switch valves. The
actuated carrier valve 40 operates the preparation loop. The
actuated sample valve 50 operates the sample loop.
[0090] The same or different carrier liquids can be maintained in
separate carrier reservoirs 70. The analyte sample, which may
contain the same or different carrier liquid as in the pumps, can
be injected into the flowing carrier using the sample injector 60.
The flowing carrier can carry the sample into the column 90, which
is packed with a particle stationary phase suitable for
chromatographic retention and analyte separation. The carrier
brings the separated analytes to the detector device 80 for
analysis. The detector device 80 can detect the separated analyte
fractions.
[0091] Referring to FIG. 2, an embodiment of a detector device 80
of this invention can include a flash lamp 110, light fibers 120, a
flow cell 130, and a UV-Vis light sensor 140. The UV-Vis light
sensor 140 can include an entrance slit 150, a grating 160, and a
CCD array 170.
[0092] An embodiment of a liquid chromatography apparatus of this
invention can also include a microcomputer controller that can
synchronize the operation of the flash lamp to the data acquisition
of the light sensor.
[0093] With this arrangement, a liquid chromatography apparatus of
this invention can provide a high light intensity for detecting
analytes. Further, constant pumping speed can be maintained.
[0094] A liquid chromatography apparatus of this invention can
advantageously use commercial chromatography columns.
[0095] A liquid chromatography apparatus of this invention can
advantageously be used in the field to for detecting analytes,
chemicals, contaminants or other species at their source.
[0096] This invention provides the advantageous result that even
with reduced mass, a liquid chromatography apparatus of this
invention can provide retention times for analytes that remain
surprisingly constant. This invention can provide surprisingly
accurate quantitative measurements even with reduced mass.
[0097] In some embodiments, the carrier tubes in the apparatus were
1/16'' and 1/32'' outer-diameter (OD), 150 micrometer (um)
inner-diameter (ID) Polytetrafluoroethylene (PTFE) tubes in channel
A and 25 um ID PTFE tubes in channel B, preparation loop.
[0098] A six-direction switch valve, for example, C52-1006I
(Valco), could be used for each of the preparation and sample
loops.
[0099] Example pressure transducers include DF2-SS-01-10K (DJ
instruments).
[0100] Example flow sensors include SLG1430 (CMOSens
Technology).
[0101] In certain embodiments, an HPLC apparatus of this invention
can be compatible with 1/16'' or 1/32'' or 380 um OD HPLC
columns.
[0102] In further aspects, an apparatus of this disclosure can have
the ability to separate various unknown samples by replacing the
column with one or more different columns.
[0103] In certain embodiments, a detector device can include a scan
range of from 230 nm to 800 nm.
[0104] In further embodiments, an HPLC apparatus of this invention
can be connected to a mass spectrometer, a nuclear magnetic
resonance spectrometer, or a light scattering detector.
[0105] In one embodiment, an HPLC apparatus of this invention was
made having a mass of less than 12 kg and a volume of less than 22
L. This apparatus had overall power consumption of less than 200 W
and was operated using a lithium-ion battery. The apparatus can
optionally be powered with conventional electrical power
sources.
[0106] In one embodiment, a detector device for an HPLC apparatus
of this invention was made having power consumption of 2 W and
overall size of 0.486 liter (9 cm by 18 cm by 3 cm).
[0107] A liquid chromatography apparatus of this invention can
advantageously provide high-throughput screening and detecting of
analytes in combination with an efficient, small volume mass
spectrometer. When the power consumption and volume of both the LC
apparatus and the mass spectrometer apparatus are reduced, the
combination is capable of high throughput detection of analytes as
compared to a conventional LC-MS apparatus.
[0108] Further, a liquid chromatography apparatus of this invention
can advantageously be used in a patient clinic.
[0109] Also, a liquid chromatography apparatus of this invention
can advantageously be used for environmental measurements in the
field or at remote locations to detect analytes, chemicals,
contaminants or other species at their source.
Example 1
[0110] An HPLC of this invention was assembled having a UV-Vis
detector device. The HPLC had less than 10 L total volume. This
HPLC was operated with less than 150 Watt overall power
consumption. Referring to FIG. 3, a chromatogram showing retention
time versus absorbance was obtained with this HPLC for a sample of
MyoGlobin. Absorbance was obtained at 400 nm wavelength.
[0111] All publications and patents and literature specifically
mentioned herein are incorporated by reference for all
purposes.
[0112] It is understood that this invention is not limited to the
particular methodology, protocols, materials, and reagents
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention, which will be encompassed by the appended
claims.
[0113] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprises," "comprising", "containing," "including", and "having"
can be used interchangeably.
[0114] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever.
[0115] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose.
* * * * *